The greatest challenge in the biosensor industry is to detect in a meaningful commercially viable manner assay reactions which take place at the molecular level. There are many situations where the detection of analyte in sample at concentrations in the picomolar range may be indicative of a particular condition in microorganisms and higher life forms, including of course animals and humans. Such demands have driven the biosensor industry towards very sophisticated and elaborate assay schemes to achieve such minute detections. However, such devices are normally too expensive to manufacture, sell and use and hence are not readily received by the research and health care communities.
Various assays which involve amplification of the signal have been effective in sensing picomolar concentrations. However, such amplification involves fairly elaborate chemistry, several sample handling and washing steps and very sophisticated sensing technology such as fluorometric detection. An example of such fluorometric amplification is described in U.S. Pat. No. 5,262,299 entitled Enzyme-Amplified Lanthanide Chelate Luminescence.
Other attempts to achieve an amplified signal indicating presence of analyte, involves the use of particles, normally polymeric, gold, solid and porous glass particles of the micron and sub-micron size. Detection based on the presence of such particles involves the use of filters, capacitance, fluorescence, magnetic separation and the like. In order to achieve the desired sensitivities in using such particles in biosensors it has been a requirement that the operation of the test device be closely controlled. Quite often the sensitivities in using such particles in biosensors it has been a requirement that the operation of the test device be closely controlled. Quite often the sensitivities in these various types of biosensors are greatly altered by minor external changes such as in temperature, pressure, flow rates, sample introduction and washing times and the like. Even with special care in providing instrument stability it is still not possible to detect changes at the molecular level where just a few molecules are detected.
Phosphorescent properties have been sensed in various types of assays, particularly with the use of metallo-porphyrins. Such compounds are encapsulated in liposomal vesicles or entrapped in polymer latex particles and bound adhesively or covalently to antibodies. The significant disadvantage in the use of porphyrin based phosphors is that the phosphorescence is quenched by oxygen in aqueous media. An attempt to eliminate this quenching is desired in U.S. Pat. No. 5,464,741 where a complex formation with the porphyrin is made.
U.S. Pat. No. 4,219,335 describes the use of reactance tags in form of small particles which by virtue of capacitance measurement provides a determination of whether or not analyte is present in the sample. The system is particularly adapted to the use of magnetic particles where the presence of the magnetic particles is measured as a change in inductance.
A more sophisticated capacitance type measurement is described in U.S. Pat. No. 4,822,566. Particles however are not used as labelling entities. Instead, the presence or absence of the complexed antibodies is detected by way of a capacitance type measurement to determine analyte concentration. However, such system is not reliable from a sensitivity standpoint and must be calibrated for each different analyte to be measured. In addition, liquid handling in this system becomes crucial.
The test system and particles used in the biosensor of this invention overcomes a number of the above problems and at the same time provides an assay system which is sensitive at the sub-picomolar or less range. Experience with current high volume automated instruments in the diagnostic field has demonstrated that 70 to 90% of the robotic cost, complexity, repeat testing, and electromechanical breakdown is related to liquid handling. The system of this invention may be reagentless or reagents-contained in the assay device in which only one liquid handling step is required during introduction of the sample to the device. The device includes a test surface which readily lends itself to simplified robotics.